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Anti-inflammatory properties of Bridelia ferruginea stem bark
Journal of Ethnopharmacology 88 (2003) 221–224
Anti-inflammatory properties of Bridelia ferruginea stem bark Inhibition of lipopolysaccaride-induced septic shock and vascular permeability Olumayokun A. Olajide∗ , David T. Okpako, Janet M. Makinde Department of Pharmacology and Therapeutics, College of Medicine, University of Ibadan, Ibadan, Nigeria Received 4 December 2002; accepted 19 June 2003
Abstract The anti-inflammatory activity of the aqueous extract of Bridelia ferruginea stem bark was further evaluated in models which are mediated by tumour necrosis factor-alpha (TNF␣). The effect of the extract on lipopolysaccharide (LPS)-induced septic shock was evaluated by measuring the number of deaths and the levels of serum alanine and aspartate aminotransferases following intraperitoneal injection of LPS (1 g/kg) into d-galactosamine-primed mice. LPS-induced vascular permeability on the back skin of mice was measured by the local accumulation of Evan’s blue after subcutaneous injection of LPS. Pre-treatment with Bridelia ferruginea extract (10–80 mg/kg) produced a dose-dependent inhibition of the septic shock syndrome in mice, with 80 mg/kg of the extract exhibiting comparable activity as pentoxifylline (100 mg/kg). LPS-induced dye leakage in the skin of mice was also suppressed by the extract (10–80 mg/kg). Our study suggests that one of the mechanisms of the anti-inflammatory effects of Bridelia ferruginea possibly involve the suppression of TNF␣ up-regulation. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Bridelia ferruginea; Anti-inflammatory; Tumour necrosis factor-alpha; Lipopolysaccharide; Septic shock; Microvascular permeability
1. Introduction Bridelia ferruginea Benth. Euphorbiaceae is a shrub, which is employed locally in arthritis, and as an embrocation for the treatment of bruises, boils, dislocation and burns (Oliver-Bever, 1986; Iwu, 1993). The plant is also used as an ingredient in some varieties of the Yoruba infusion called ‘agbo’ in paediatric illnesses, especially malaria fever (Iwu, 1993). Earlier, we observed that the aqueous extract of Bridelia ferruginea stem bark exhibited inhibition of both the carrageenan-induced rat paw oedema and cotton pellet granuloma formation in rats (Olajide et al., 1999). Topical anti-inflammatory, anti-arthritic, antipyretic and analgesic properties of the plant were also reported by us (Olajide et al., 2000). Chemical examination of the leaf of Bridelia ferruginea resulted in the isolation and characterization of bridelilactone and bridelilactoside, which are coumenstaflavonoids (Iwu, 1993). Flavonoids and biflavonoids based on apigenin and kaempferol moieties were also isolated together with ∗ Corresponding
author. E-mail address: [email protected] (O.A. Olajide).
their glycosides from the methanolic extract of the plant (Iwu, 1993). The flavonoids gallocatechin-(4-O-7-epigallocatechin), quercetin-3,3-methylether, 3,5-dicaffeoylquinic acid, quercetin 3,7,3,4-tetramethylether, myricetin and quercetin 3-O-glucoside have been isolated from extracts of Bridelia ferruginea (Cimanga et al., 1999, 2001; De-Bruyne et al., 1998). In this study, the anti-inflammatory profile of the aqueous extract of Bridelia ferruginea stem bark was further evaluated in both LPS-induced septic shock and LPS-induced microvascular permeability. The pro-inflammatory cytokinetumour necrosis factor-alpha (TNF␣), has been shown to be one of the endogenous chemicals, which mediate the inflammatory response in these models. They were therefore employed in this study to elucidate the possible roles of TNF␣ in the anti-inflammatory activity of the plant.
2. Materials and methods 2.1. Animals Male Swiss mice (22–25 g) were used for this study. The animals were bred and housed in the Pre-Clinical Animal
0378-8741/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00219-8
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O.A. Olajide et al. / Journal of Ethnopharmacology 88 (2003) 221–224
House, College of Medicine, University of Ibadan, which was well-ventilated. The animals were fed on standard diet (Ladokun Feeds, Ibadan) and water ad libitum. 2.2. Plant materials Bridelia ferruginea stem barks were collected in the month of May 1998 from a tree growing along Amina Way, University of Ibadan Campus, Ibadan, South West Nigeria. The plant samples were identified in the Herbarium, Botany Department, University of Ibadan as well as the Herbarium, Forestry Research Institute of Nigeria (FRIN). These samples were authenticated using voucher specimens deposited at various periods. A voucher specimen of the collected plant samples was also deposited in the FRIN herbarium, and given the Specimen Number F.H.I. 106501. The stem barks were air-dried at room temperature, powdered and extracted in distilled water for 18 h. The extract obtained was concentrated to a solid-brown residue. The yield of the aqueous extract was 10.1%, based on starting material (stem bark). The extract obtained was stored in a refrigerator. A stock solution of 100 mg/ml of the extract was prepared in 0.9% normal saline for pharmacological studies.
eighth hour), the number of animals that died was recorded, and blood was quickly withdrawn by cardiac puncture as soon as the animals lapsed into paralysis, just before death. The blood samples from each mouse was collected into tubes, and the serum levels of both alanine and aspartate aminotransferases were determined using the method of Reitman and Frankel (1957). 2.4. Effect of the extract on plasma leakage in mouse skin This was done using the method earlier described by Irie et al. (2001), with slight modifications. The microvascular permeability of the skin was assessed by an extravasation of Evan’s blue. The dye (100 mg/kg) was injected to the mouse via the tail vein, and 5 min later, LPS (400 g/site) was subcutaneously administered at the back of the mouse. Two hours later, the mice were killed by cervical dislocation and stained area of the skin at the site of injection was excised (about 1 g) and minced. The skin specimen was dispersed in 6 ml 0.5% Na2 SO4 and the dye was extracted by an addition of 14 ml acetone. After 3.5 h of extraction period, the dye concentration was determined by a spectrophotometer at 590 nm. The extracts (10–80 mg/kg) and pentoxifylline (100 mg/kg) were administered 1 h prior to Evan’s blue injection.
2.3. Effect of the extract on LPS-induced septic shock 2.5. Statistical analysis The experiment was carried out as described by Gantner et al. (1995), with slight modifications. Mice were divided into seven treatment groups with six animals in a group (n = 6). Groups of mice were administered intraperitoneally with 10, 20, 40 and 80 mg/kg of Bridelia ferruginea extract. The positive control group was given pentoxifylline (100 mg/kg, i.p.). Animals in the control group were administered saline at a dose of 10 ml/kg. Thirty minutes later, all the animals were injected with d-galactosamine (700 mg/kg, i.p.), followed by LPS (1 g/kg, i.p.). The negative control group was injected only with d-galactosamine and saline. Eight hours later, blood was collected from all the mice by cardiac puncture following lethal anaesthesia with 150 mg/kg pentobarbital. In cases where animals died before the termination of the experiment (that is, before the
All data were expressed as means±S.E.M. Statistical significance was determined using the Student’s t-test. Values with P < 0.05 were considered significant.
3. Results 3.1. Effect of the extract on LPS-induced septic shock Three parameters were measured in this experiment: number of deaths per group, serum levels of alanine and aspartate aminotransferases (Table 1). No deaths were recorded in the group of mice given only saline and d-galactosamine. However, in galactosamine-primed mice injected with LPS, all
Table 1 Effect of Bridelia ferruginea stem bark aqueous extract on LPS-induced septic shock in mice Treatment
Number of deaths
Serum enzyme activity (U/ml)a ALT
Saline + DG Saline + DG + LPS Bridelia ferruginea (10 mg/kg) + DG + LPS Bridelia ferruginea (20 mg/kg) + DG + LPS Bridelia ferruginea (40 mg/kg) + DG + LPS Bridelia ferruginea (80 mg/kg) + DG + LPS Pentoxifylline (100 mg/kg) + DG + LPS a b
0/6 6/6 3/6 2/6 1/6 0/6 0/6
Each value is the mean ± S.E.M. of six animals. P < 0.05 compared with DG + saline + LPS treatment; Student’s t-test DG-d-galactosamine.
270.7 297.8 284.0 278.2 274.3 266.3 271.0
AST ± ± ± ± ± ± ±
3.2 3.5 5.5 4.2b 1.9b 2.4b 4.2b
304.5 335.8 315.5 308.7 304.0 290.5 309.5
± ± ± ± ± ± ±
6.6 7.8 6.5b 7.4b 5.6b 3.3b 6.7b
O.A. Olajide et al. / Journal of Ethnopharmacology 88 (2003) 221–224 Table 2 Effect of Bridelia ferruginea stem bark aqueous extract on LPS-induced microvascular permeability in mice Treatment
Amount of dye leaked (g/g wet weight)a
Saline + LPS Bridelia ferruginea (10 mg/kg) + LPS Bridelia ferruginea (20 mg/kg) + LPS Bridelia ferruginea (40 mg/kg) + LPS Bridelia ferruginea (80 mg/kg) + LPS Pentoxifylline (100 mg/kg) + LPS
57.3 38.0 28.3 19.3 12.7 12.8
a b
± ± ± ± ± ±
4.9 2.8b 1.9b 1.1b 1.2b 1.4b
Each value is the mean ± S.E.M. of six animals. P < 0.05 compared with saline + LPS treatment; Student’s t-test.
the animals died before the termination of the experiment. The number of deaths in the groups of animals pre-treated with the extract of Bridelia ferruginea prior to LPS challenge was three, two, one none for 10, 20,40 and 80 mg/kg of the extract, respectively. No death was also recorded in the group of mice that received pentoxifylline (100 mg/kg). d-Galactosamine injection before saline treatment resulted in the serum levels of 270.7 ± 3.2 U/ml and 304.5±6.6 U/ml of alanine and aspartate aminotransferases, respectively. In d-galactosamine-primed mice injected with LPS, the levels were elevated to 297.8 ± 3.5 U/ml and 335.8 ± 7.8 U/ml for alanine and aspartate aminotransferases, respectively. Pre-treatment with Bridelia ferruginea extract, however, caused a statistically significant (P < 0.05) reduction in the levels of both enzymes. Pentoxifylline pre-treatment also caused a significant (P < 0.05) reduction in the serum enzyme levels. 3.2. Effect of the extract on plasma leakage in mouse skin The effects of the extracts on LPS-induced dye leakage were evaluated 2 h after subcutaneous injection of LPS. Pre-treatment with the Bridelia ferruginea extract produced a dose-related inhibition of dye leakage (Table 2). Dye leakage in mice given only saline before LPS injection was 57.3 ± 4.9 g/g wet weight. This was significantly (P < 0.05) reduced to 38.0 ± 2.8 g/g wet weight by the extract (10 mg/kg). The highest dose of the extract used in this study (80 mg/kg) inhibited dye leakage to a similar degree as pentoxifylline (100 mg/kg).
4. Discussion The extract of Bridelia ferruginea protected mice from septic shock in a dose-dependent fashion. The serum levels of liver enzymes were also reduced in mice treated with the extract. There was no death recorded in mice given only saline and d-galactosamine. This shows that the death of animals primed with d-galactosamine and injected with LPS was not due to the action of the hepatotoxic agent.
223
LPS/d-galactosamine-induced sepsis has been reported to be mediated by TNF␣ (Dinarello, 1997). In addition, TNF␣ has been demonstrated to mediate LPS-induced systemic inflammatory response syndrome, that is liver failure, characterised by early apoptosis and subsequent liver cell death (Tiegs et al., 1989; Leist et al., 1995). TNF␣ activity in this model has been shown by Gantner et al. (1995) to correlate with elevated levels of transaminase enzymes and septic death. Pentoxifylline was used as a reference agent in this study. The drug is a class IV phosphodieserase inhibitor which has been shown to be TNF␣ antagonist in animal experiments (Le May et al., 1990; Goldbach et al., 1997). Other class IV phosphodiesterase inhibitors like rolipram and compound CP-77059 were shown by Sekut et al. (1995) to exert anti-inflammatory activities in different murine models. The effects of Bridelia ferruginea extract was also tested on LPS-induced dye leakage in the mouse skin. The effects of the extract and pentoxifylline were evaluated 2 h after injection of LPS, as Fujii et al. (1996) have shown that the dye leakage by LPS in the mouse skin reached a maximum at 2 h. The extract was found to produce inhibition of dye leakage to the same degree as pentoxifylline. Subcutaneous injection of LPS on the back of mice and rats induces a plasma leakage at the site of injection, and this is used as a model of inflammation (Fujii et al., 2000; Wada et al., 2000). The LPS-induced increase in vascular permeability has been reported to be mediated by TNF␣ and nitric oxide among other mediators in the mouse (Wada et al., 2000). This study has therefore provided an evidence for a possible role for TNF␣ in the anti-inflammatory effects of the stem bark extract of Bridelia ferruginea. It is yet to be determined if other cytokines, like interleukin-1, play any role in this activity of the plant. Further studies on the effects of the plant on TNF␣ and nitric oxide production from cells, as well as its effects on the gene expressions of these mediators would be necessary.
References Cimanga, K., De-Bruyne, T., Apers, S., Pieters, L., Totte, M.J., Kambu, K., Tona, L., Bakana, P., van Ufford, L.Q., Beukelman, C., Labadie, R., Vlietinck, A.J., 1999. Complement-inhibiting constituents of Bridelia ferruginea stem bark. Planta Medica 65, 213–217. Cimanga, K., Ying, L., De-Bruyne, T., Apers, S., Cos, P., Hermans, N., Bakana, P., Tona, L., Kambu, K., Kalenda, D.T., Pieters, L., Berghe, D.V., Vlientick, A.J., 2001. Radical scavenging and xanthine oxidase inhibitory activity of phenolic compounds from Bridelia ferruginea stem bark. Journal of Pharmacy and Pharmacology 53, 757–761. De-Bruyne, T., Cimanga, K., Pieters, L., Claeys, M., Dommisse, R., Vlientick, A.J., 1998. Gallocatechin-(4-0-7)-epigallocatechin, a new biflavonoid isolated from Bridelia ferruginea. Natural Products Letters 11, 47–52. Dinarello, C.A., 1997. Role of pro- and anti-inflammatory cytokines during inflammation: experimental and clinical findings. Journal of Biological Regulators and Homeostatic Agents 11, 91–103. Fujii, E., Irie, K., Ogawa, A., Ohba, K., Muraki, T., 1996. Role of nitric oxide and prostaglandins in lipopolysaccharide-induced increase
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in vascular permeability in mouse skin. European Journal of Pharmacology 297, L675–L682. Fujii, E., Yoshioka, T., Ishida, H., Irie, K., Muraki, T., 2000. Evaluation of iNOS dependent and independent mechanisms of the microvascular permeability change induced by lipopolysaccharide. British Journal of Pharmacology 130, 90–94. Gantner, F., Uhlig, S., Wendel, A., 1995. Quinine inhibits release of tumour necrosis factor, apoptosis, necrosis and mortality in a murine model of septic liver failure. European Journal of Pharmacology 29, 353–355. Goldbach, J.M., Roth, J., Storr, B., Zeisberger, E., 1997. Influence of pentoxifylline on fevers induced by bacterial lipopolysaccharide and tumour necrosis factor-␣ in guinea pigs. European Journal of Pharmacology 319, 273–278. Irie, K., Fujii, E., Ishida, H., Wada, K., Suganuma, T., Nishikori, T., Yoshioka, T., Muraki, T., 2001. Inhibitory effects of cyclic AMP elevating agents on lipopolysaccharide (LPS)-induced microvascular permeability change in mouse skin. British Journal of Pharmacology 133, 237–242. Iwu, M.M., 1993. Handbook of African Medicinal Plants. CRC Press, Boca Raton, FL, pp. 133–135. Le May, L.G., Vander, J., Kluger, M.J., 1990. The effects of pentoxifylline on lipopolysaccharide (LPS) fever, plasma interleukin-6 (IL-6) and tumour necrosis factor (TNF) in the rat. Cytokine 2, 300. Leist, M.F., Gantner, F., Bohlinger, I., Tiegs, G., Germann, P.G., Wendel, A., 1995. Tumour necrosis factor-induced hepatocyte apoptosis
precedes liver failure in experimental murine shock models. American Journal of Pathology 146, 1220. Olajide, O.A., Makinde, J.M., Awe, S.O., 1999. Effects of the aqueous extract of Bridelia ferruginea stem bark on carrageenan-induced oedema and granuloma formation in rats and mice. Journal of Ethnopharmacology 66, 113–117. Olajide, O.A., Makinde, J.M., Okpako, D.T., Awe, S.O., 2000. Studies on the anti-inflammatory and related pharmacological properties of the aqueous extract of Bridelia ferruginea stem bark. Journal of Ethnopharmacology 71, 153–160. Oliver-Bever, B., 1986. Medicinal Plants in Tropical West Africa. Cambridge University Press, London, p. 260. Reitman, S., Frankel, S., 1957. A colorimetric method for the determination of serum GOT and GPT. American Journal of Clinical Pathology 28, 56–63. Sekut, L., Yarnall, D., Stimpson, S.A., Noel, L.S., Bateman-Fite, R., Clark, R.L., Brackeen, M.F., Menius, J.A., Connolly, K.M., 1995. Anti-inflammatory activity of phosphodiesterase (PDE)-IV inhibitors in acute and chronic models of inflammation. Clinical and Experimental Immunology 100, 126–132. Tiegs, G., Wolter, M., Wendel, A., 1989. Tumour necrosis factor is a terminal mediator in d-galactosamine endotoxin-induced hepatitis in mice. Biochemical Pharmacology 38, 627. Wada, K., Fujii, E., Ishida, H., Yoshioka, T., Muraki, T., 2000. Effect of lipoteichoic acid on dermal vascular permeability in mice. Journal of Pharmacology and Experimental Therapeutics 294, 280–286.
Anti-inflammatory properties of Bridelia ferruginea stem bark Inhibition of lipopolysaccaride-induced septic shock and vascular permeability Olumayokun A. Olajide∗ , David T. Okpako, Janet M. Makinde Department of Pharmacology and Therapeutics, College of Medicine, University of Ibadan, Ibadan, Nigeria Received 4 December 2002; accepted 19 June 2003
Abstract The anti-inflammatory activity of the aqueous extract of Bridelia ferruginea stem bark was further evaluated in models which are mediated by tumour necrosis factor-alpha (TNF␣). The effect of the extract on lipopolysaccharide (LPS)-induced septic shock was evaluated by measuring the number of deaths and the levels of serum alanine and aspartate aminotransferases following intraperitoneal injection of LPS (1 g/kg) into d-galactosamine-primed mice. LPS-induced vascular permeability on the back skin of mice was measured by the local accumulation of Evan’s blue after subcutaneous injection of LPS. Pre-treatment with Bridelia ferruginea extract (10–80 mg/kg) produced a dose-dependent inhibition of the septic shock syndrome in mice, with 80 mg/kg of the extract exhibiting comparable activity as pentoxifylline (100 mg/kg). LPS-induced dye leakage in the skin of mice was also suppressed by the extract (10–80 mg/kg). Our study suggests that one of the mechanisms of the anti-inflammatory effects of Bridelia ferruginea possibly involve the suppression of TNF␣ up-regulation. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Bridelia ferruginea; Anti-inflammatory; Tumour necrosis factor-alpha; Lipopolysaccharide; Septic shock; Microvascular permeability
1. Introduction Bridelia ferruginea Benth. Euphorbiaceae is a shrub, which is employed locally in arthritis, and as an embrocation for the treatment of bruises, boils, dislocation and burns (Oliver-Bever, 1986; Iwu, 1993). The plant is also used as an ingredient in some varieties of the Yoruba infusion called ‘agbo’ in paediatric illnesses, especially malaria fever (Iwu, 1993). Earlier, we observed that the aqueous extract of Bridelia ferruginea stem bark exhibited inhibition of both the carrageenan-induced rat paw oedema and cotton pellet granuloma formation in rats (Olajide et al., 1999). Topical anti-inflammatory, anti-arthritic, antipyretic and analgesic properties of the plant were also reported by us (Olajide et al., 2000). Chemical examination of the leaf of Bridelia ferruginea resulted in the isolation and characterization of bridelilactone and bridelilactoside, which are coumenstaflavonoids (Iwu, 1993). Flavonoids and biflavonoids based on apigenin and kaempferol moieties were also isolated together with ∗ Corresponding
author. E-mail address: [email protected] (O.A. Olajide).
their glycosides from the methanolic extract of the plant (Iwu, 1993). The flavonoids gallocatechin-(4-O-7-epigallocatechin), quercetin-3,3-methylether, 3,5-dicaffeoylquinic acid, quercetin 3,7,3,4-tetramethylether, myricetin and quercetin 3-O-glucoside have been isolated from extracts of Bridelia ferruginea (Cimanga et al., 1999, 2001; De-Bruyne et al., 1998). In this study, the anti-inflammatory profile of the aqueous extract of Bridelia ferruginea stem bark was further evaluated in both LPS-induced septic shock and LPS-induced microvascular permeability. The pro-inflammatory cytokinetumour necrosis factor-alpha (TNF␣), has been shown to be one of the endogenous chemicals, which mediate the inflammatory response in these models. They were therefore employed in this study to elucidate the possible roles of TNF␣ in the anti-inflammatory activity of the plant.
2. Materials and methods 2.1. Animals Male Swiss mice (22–25 g) were used for this study. The animals were bred and housed in the Pre-Clinical Animal
0378-8741/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00219-8
222
O.A. Olajide et al. / Journal of Ethnopharmacology 88 (2003) 221–224
House, College of Medicine, University of Ibadan, which was well-ventilated. The animals were fed on standard diet (Ladokun Feeds, Ibadan) and water ad libitum. 2.2. Plant materials Bridelia ferruginea stem barks were collected in the month of May 1998 from a tree growing along Amina Way, University of Ibadan Campus, Ibadan, South West Nigeria. The plant samples were identified in the Herbarium, Botany Department, University of Ibadan as well as the Herbarium, Forestry Research Institute of Nigeria (FRIN). These samples were authenticated using voucher specimens deposited at various periods. A voucher specimen of the collected plant samples was also deposited in the FRIN herbarium, and given the Specimen Number F.H.I. 106501. The stem barks were air-dried at room temperature, powdered and extracted in distilled water for 18 h. The extract obtained was concentrated to a solid-brown residue. The yield of the aqueous extract was 10.1%, based on starting material (stem bark). The extract obtained was stored in a refrigerator. A stock solution of 100 mg/ml of the extract was prepared in 0.9% normal saline for pharmacological studies.
eighth hour), the number of animals that died was recorded, and blood was quickly withdrawn by cardiac puncture as soon as the animals lapsed into paralysis, just before death. The blood samples from each mouse was collected into tubes, and the serum levels of both alanine and aspartate aminotransferases were determined using the method of Reitman and Frankel (1957). 2.4. Effect of the extract on plasma leakage in mouse skin This was done using the method earlier described by Irie et al. (2001), with slight modifications. The microvascular permeability of the skin was assessed by an extravasation of Evan’s blue. The dye (100 mg/kg) was injected to the mouse via the tail vein, and 5 min later, LPS (400 g/site) was subcutaneously administered at the back of the mouse. Two hours later, the mice were killed by cervical dislocation and stained area of the skin at the site of injection was excised (about 1 g) and minced. The skin specimen was dispersed in 6 ml 0.5% Na2 SO4 and the dye was extracted by an addition of 14 ml acetone. After 3.5 h of extraction period, the dye concentration was determined by a spectrophotometer at 590 nm. The extracts (10–80 mg/kg) and pentoxifylline (100 mg/kg) were administered 1 h prior to Evan’s blue injection.
2.3. Effect of the extract on LPS-induced septic shock 2.5. Statistical analysis The experiment was carried out as described by Gantner et al. (1995), with slight modifications. Mice were divided into seven treatment groups with six animals in a group (n = 6). Groups of mice were administered intraperitoneally with 10, 20, 40 and 80 mg/kg of Bridelia ferruginea extract. The positive control group was given pentoxifylline (100 mg/kg, i.p.). Animals in the control group were administered saline at a dose of 10 ml/kg. Thirty minutes later, all the animals were injected with d-galactosamine (700 mg/kg, i.p.), followed by LPS (1 g/kg, i.p.). The negative control group was injected only with d-galactosamine and saline. Eight hours later, blood was collected from all the mice by cardiac puncture following lethal anaesthesia with 150 mg/kg pentobarbital. In cases where animals died before the termination of the experiment (that is, before the
All data were expressed as means±S.E.M. Statistical significance was determined using the Student’s t-test. Values with P < 0.05 were considered significant.
3. Results 3.1. Effect of the extract on LPS-induced septic shock Three parameters were measured in this experiment: number of deaths per group, serum levels of alanine and aspartate aminotransferases (Table 1). No deaths were recorded in the group of mice given only saline and d-galactosamine. However, in galactosamine-primed mice injected with LPS, all
Table 1 Effect of Bridelia ferruginea stem bark aqueous extract on LPS-induced septic shock in mice Treatment
Number of deaths
Serum enzyme activity (U/ml)a ALT
Saline + DG Saline + DG + LPS Bridelia ferruginea (10 mg/kg) + DG + LPS Bridelia ferruginea (20 mg/kg) + DG + LPS Bridelia ferruginea (40 mg/kg) + DG + LPS Bridelia ferruginea (80 mg/kg) + DG + LPS Pentoxifylline (100 mg/kg) + DG + LPS a b
0/6 6/6 3/6 2/6 1/6 0/6 0/6
Each value is the mean ± S.E.M. of six animals. P < 0.05 compared with DG + saline + LPS treatment; Student’s t-test DG-d-galactosamine.
270.7 297.8 284.0 278.2 274.3 266.3 271.0
AST ± ± ± ± ± ± ±
3.2 3.5 5.5 4.2b 1.9b 2.4b 4.2b
304.5 335.8 315.5 308.7 304.0 290.5 309.5
± ± ± ± ± ± ±
6.6 7.8 6.5b 7.4b 5.6b 3.3b 6.7b
O.A. Olajide et al. / Journal of Ethnopharmacology 88 (2003) 221–224 Table 2 Effect of Bridelia ferruginea stem bark aqueous extract on LPS-induced microvascular permeability in mice Treatment
Amount of dye leaked (g/g wet weight)a
Saline + LPS Bridelia ferruginea (10 mg/kg) + LPS Bridelia ferruginea (20 mg/kg) + LPS Bridelia ferruginea (40 mg/kg) + LPS Bridelia ferruginea (80 mg/kg) + LPS Pentoxifylline (100 mg/kg) + LPS
57.3 38.0 28.3 19.3 12.7 12.8
a b
± ± ± ± ± ±
4.9 2.8b 1.9b 1.1b 1.2b 1.4b
Each value is the mean ± S.E.M. of six animals. P < 0.05 compared with saline + LPS treatment; Student’s t-test.
the animals died before the termination of the experiment. The number of deaths in the groups of animals pre-treated with the extract of Bridelia ferruginea prior to LPS challenge was three, two, one none for 10, 20,40 and 80 mg/kg of the extract, respectively. No death was also recorded in the group of mice that received pentoxifylline (100 mg/kg). d-Galactosamine injection before saline treatment resulted in the serum levels of 270.7 ± 3.2 U/ml and 304.5±6.6 U/ml of alanine and aspartate aminotransferases, respectively. In d-galactosamine-primed mice injected with LPS, the levels were elevated to 297.8 ± 3.5 U/ml and 335.8 ± 7.8 U/ml for alanine and aspartate aminotransferases, respectively. Pre-treatment with Bridelia ferruginea extract, however, caused a statistically significant (P < 0.05) reduction in the levels of both enzymes. Pentoxifylline pre-treatment also caused a significant (P < 0.05) reduction in the serum enzyme levels. 3.2. Effect of the extract on plasma leakage in mouse skin The effects of the extracts on LPS-induced dye leakage were evaluated 2 h after subcutaneous injection of LPS. Pre-treatment with the Bridelia ferruginea extract produced a dose-related inhibition of dye leakage (Table 2). Dye leakage in mice given only saline before LPS injection was 57.3 ± 4.9 g/g wet weight. This was significantly (P < 0.05) reduced to 38.0 ± 2.8 g/g wet weight by the extract (10 mg/kg). The highest dose of the extract used in this study (80 mg/kg) inhibited dye leakage to a similar degree as pentoxifylline (100 mg/kg).
4. Discussion The extract of Bridelia ferruginea protected mice from septic shock in a dose-dependent fashion. The serum levels of liver enzymes were also reduced in mice treated with the extract. There was no death recorded in mice given only saline and d-galactosamine. This shows that the death of animals primed with d-galactosamine and injected with LPS was not due to the action of the hepatotoxic agent.
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LPS/d-galactosamine-induced sepsis has been reported to be mediated by TNF␣ (Dinarello, 1997). In addition, TNF␣ has been demonstrated to mediate LPS-induced systemic inflammatory response syndrome, that is liver failure, characterised by early apoptosis and subsequent liver cell death (Tiegs et al., 1989; Leist et al., 1995). TNF␣ activity in this model has been shown by Gantner et al. (1995) to correlate with elevated levels of transaminase enzymes and septic death. Pentoxifylline was used as a reference agent in this study. The drug is a class IV phosphodieserase inhibitor which has been shown to be TNF␣ antagonist in animal experiments (Le May et al., 1990; Goldbach et al., 1997). Other class IV phosphodiesterase inhibitors like rolipram and compound CP-77059 were shown by Sekut et al. (1995) to exert anti-inflammatory activities in different murine models. The effects of Bridelia ferruginea extract was also tested on LPS-induced dye leakage in the mouse skin. The effects of the extract and pentoxifylline were evaluated 2 h after injection of LPS, as Fujii et al. (1996) have shown that the dye leakage by LPS in the mouse skin reached a maximum at 2 h. The extract was found to produce inhibition of dye leakage to the same degree as pentoxifylline. Subcutaneous injection of LPS on the back of mice and rats induces a plasma leakage at the site of injection, and this is used as a model of inflammation (Fujii et al., 2000; Wada et al., 2000). The LPS-induced increase in vascular permeability has been reported to be mediated by TNF␣ and nitric oxide among other mediators in the mouse (Wada et al., 2000). This study has therefore provided an evidence for a possible role for TNF␣ in the anti-inflammatory effects of the stem bark extract of Bridelia ferruginea. It is yet to be determined if other cytokines, like interleukin-1, play any role in this activity of the plant. Further studies on the effects of the plant on TNF␣ and nitric oxide production from cells, as well as its effects on the gene expressions of these mediators would be necessary.
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